METHOD FOR THE DIAGNOSTIC AND/OR PROGNOSTIC ASSESSMENT OF ACUTE-ON-CHRONIC LIVER FAILURE SYNDROME IN PATIENTS WITH LIVER DISORDERS

Abstract

An ex vivo method for the diagnostic and/or prognostic assessment of the acute-on-chronic liver failure (ACLF) syndrome in a patient with a liver disorder, includes measuring a panel of metabolites related with acylcarnitines-sialic acid-acetylated amino acids and amino acid derivatives and/or sugar alcohols, catecholamines and pyrimidine derivatives in a biological sample of the patient, and comparing the level of the metabolites in the sample with the level of the metabolites in healthy patients. An increase of at least 1.2 times of the level of the metabolites is indicative of ACLF syndrome.

Claims

1. An ex vivo method for diagnostic and/or prognostic and treatment of acute-on-chronic liver failure (ACLF) syndrome in a patient with a liver disorder, comprising: a) obtaining a biological sample or having a biological sample obtained from said patient; b) measuring or having measured a level of each of a panel of metabolites selected from the group consisting of acylcarnites, sialic acid, acetylated amino acids and amino acid derivatives and/or sugar alcohols, catecholamines and nucleoside derivatives in the biological sample from the patient; c) comparing the measured level of each of said metabolites in the sample with a level of each of said metabolites in healthy patients; and d) determining that the level of a plurality of said metabolites is increased by at least 1.2 times in the patient compared to the level of the plurality of metabolites in the healthy patients, thereby determining that the patient has or is likely to develop the ACLF syndrome: e) stratifying the patient by prognosis and based on said prognosis providing a treatment selected from the group consisting of: i) intensive care treatment alone; ii) emergency transplantation; iii) rengenerative therapy; and iv) bioartificial liver support.

2. The method according to claim 1, wherein said acylcarnitines, sialic acid, acetylated amino acids and amino acid derivatives are selected from the group consisting of 2-Heptanone, cystathionine, hexanoylcarnitine, N-Acetylneuraminic acid, N-Acetyl-L-phenylalanine, 2,2′-Thiodiacetic acid, octanoylcarnitine, trisaccharides, hydroxyphenylacetic acids, N6,N6,N6-Trimethyl-L-lysine, N-Acetyl-L-alanine, N-Acetyl-Asp-Glu, N-Acetyl-L-tyrosine, butyrylcarnitine, p-Hydroxyphenyllactic acid, phenylllactic acid, N-Acetyl-L-aspartic acid, L-Saccharopine, Methylimidazoleacetic acid and N-Formyl-L-methionine.

3. The method according to claim 1, wherein said sugar alcohols, catecholamines and nucleoside derivatives are selected from the group consisting of pentose phosphates, pentose alcohols, hexose alcohols, D-Galacturonic acid, beta-Pseudouridine, D-glucuronic acid, 4-Hydroxy-3-methoxyphenylglycol sulfate, D-Threitol, mevalonic acid, orotidine, 5′Deoxy-5′-(methylthio)adenosine, and Guanidinosuccinic acid.

4. The method according to claim 1, wherein said biological sample is saliva, whole blood, plasma, serum, or urine of a patient.

5. The method according to claim 4, wherein said biological sample is plasma or serum.

6. The method according to claim 1, wherein said measured level of said metabolites predicts the development of organ failure.

7. The method according to claim 6, wherein said organ is liver, brain or kidney.

8. The method according to claim 6, wherein an increased level of pentose phosphates, 2-Heptanone, N-Formyl-L-methionine, D-Threitol, N-Acetyl-L-aspartic acid, D-Galacturonic acid or p-Hydroxyphenyllactic acid is indicative of liver failure.

9. The method according to claim 6, wherein an increased level of Methylimidazoleacetic acid, Guanidinosuccinic acid, N-Formyl-L-methionine, N-Acetyl-L-tyrosine or N-Acetyl-L-aspartic acid is indicative of brain failure.

10. The method according to claim 6, wherein an increased level of 2-Heptanone, Trisaccharides, Cystathionine, Mevalonic acid, Octanoylcarnitine or p-Hydroxyphenyllactic acid is indicative of kidney failure.

Description

EXAMPLES

Example 1: Characterization of the Patients with Acutely Decompensated Cirrhosis whose Biobanked Serum Samples have been used for Metabolomics.

[0031] The study used biobanked serum samples from patients with acutely decompensated cirrhosis (334 with AD (without ACLF); 179 with ACLF) prospectively enrolled in the CANONIC study, along with 20 patients with compensated cirrhosis, and 29 healthy subjects. All these individuals or their legal representatives and the ethics committee of each hospital involved in the study gave informed consent for omics investigations in the biobanked material. Values for physiological variables and plasma cytokines and chemokines, neutrophil gelatinase-associated lipocalin (NGAL), soluble CD206 and soluble CD163 given in the manuscript were among those previously reported in other studies. The clinical and standard laboratory characteristics of the patients included in the current investigation are illustrated in Table 1.

[0032] Samples of serum from patients with decompensated cirrhosis with and without ACLF were analyzed by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Two complementary methods involving both HILIC and C18 columns for metabolomics and C8 column for lipidomics were used in both positive and negative ionization modes (Boudah et al, J Chrom B 2014; Seyer et al, Metabolomics 2016).

TABLE-US-00001 TABLE 1 Characteristics of the AD and ACLF patients. AD ACLF P ACLF-1 ACLF-2 ACLF-3 P- Characteristic (n = 334) (n = 179) value (n = 95) (n = 65) (n = 19) value Demographical and clinical data Age, y, mean ± SD 57.1 ± 11.5 56.9 ± 11.3 0.85 58.7 ± 11.5 55.5 ± 10.9 52.6 ± −9.9 0.05 Male sex, n (%) 221 (66.2) 117 (65.4) 0.85 65 (68.4) 43 (66.1) 9 (47.4) 0.21 Ascites, n (%) 187 (56.2) 130 (73.0) <0.01 64 (67.4) 51 (79.7) 15 (78.9) 0.19 Potential precipitating events of ACLF, n (%) Bacterial Infection 77 (23.1) 62 (35.0) <0.01 28 (29.8) 22 (34.4) 12 (63.2) 0.02 Active alcoholism 37 (11.1) 32 (17.9) 0.07 11 (11.6) 16 (24.6) 5 (26.3) 0.12 Other precipitating 8 (2.4) 13 (7.3) <0.01 5 (5.3) 7 (11.8) 1 (5.7) 0.39 event No precipitating 149 (44.6) 74 (43.3) 0.58 48 (50.5) 22 (33.9) 15 (78.9) 0.03 event Organ failures, n (%) Liver 36 (10.8) 71 (39.7) <0.01 21 (22.1) 36 (55.4) 14 (73.7) <0.01 Kidney 0.00 (0) 103 (57.5) <0.01 61 (64.2) 27 (41.5) 15 (78.9) <0.01 Brain 8 (2.4) 35 (19.6) <0.01 3 (3.2) 21 (32.3) 11 (57.9) <0.01 Coagulation 12 (3.6) 44 (24.6) <0.01 6 (6.3) 26 (40.0) 12 (63.2) <0.01 Circulation 4 (1.2) 29 (16.2) <0.01 1 (1.1) 14 (21.5) 14 (73.7) <0.01 Lungs 3 (0.9) 14 (7.8) <0.01 3 (3.2) 6 (9.2) 5 (26.3) <0.01 Organ dysfunctions, n (%) Liver 118 (35.3) 102 (57.0) <0.01 39 (41.0) 48 (73.9) 15 (78.9) <0.01 Kidney 60 (18.0) 118 (65.9) <0.01 72 (75.8) 32 (49.2) 14 (73.7) <0.01 Brain 88 (26.4) 104 (58.1) <0.01 52 (54.7) 36 (55.4) 16 (84.2) 0.05 Mortality, n (%) 28-day Mortality 22 (6.6) 48 (26.8) <0.01 17 (17.9) 18 (27.7) 13 (68.4) <0.01 90-day Mortality 55 (16.5) 72 (40.2) <0.01 29 (30.5) 30 (46.2) 13 (68.4) <0.01 Physiological variables Serum bilirubin, mg/dL, 5.6 ± 6.5 11.3 ± 11.4 <0.01 6.8 ± 8.0 14.2 ± 10.7 24.4 ± 14.9 <0.01 mean ± SD International 1.6 ± 0.4 2.0 ± 0.9 <0.01 1.6 ± 0.5 2.4 ± 0.9 2.9 ± 1.2 <0.01 normalized ratio, mean ± SD Median values of aminotransferases (IQR), U/L Alanine 36.0 (24-59.5) 35 (22-57) 0.71 30 (18-48) 37.5 (28-54) 58 (31-129) <0.01 aminotransferase Aspartate 59.5 (38-102) 67 (37-111) 0.47 49.5 (34-93) 76 (46-111) 122.5 (89-149) <0.01 aminotransferase Serum creatinine, mg/dL, 1.0 ± 0.4 2.3 ± 1.5 <0.01 2.5 ± 1.5 1.9 ± 1.5 2.9 ± 1.9 <0.01 mean ± SD Blood white-cell 7.4 ± 4.5 9.9 ± 7.1 <0.01 8.2 ± 4.8 11.2 ± 7.0  14.6 ± 13.2 <0.01 count, ×10.sup.9/L, mean ± SD Median levels of 107 (90-144) 108 (87-142) 0.71 107 (89-153) 107 (86-133) 116 (85-153) 0.74 blood glucose (IQR), mg/dL

Example 2: Characterization of Patient's Metabolic Profile Associated with ACLF.

[0033] The aim of the study was to characterize the metabolic profile associated with ACLF, to assess if there was a blood metabolite fingerprint specific for ACLF of any grade and each of the three ACLF grades of severity.

[0034] Among the 137 annotated metabolites identified, 32 were increased in ACLF of any grade, relative to patients with acute decompensation. These 32 metabolites compose a specific blood metabolite fingerprint. This metabolite fingerprint is useful for the diagnosis of ACLF. The prototypic metabolites identified in this fingerprint are illustrated in the following tables. These metabolites can be divided in two different subgroups:

[0035] Group 1: acylcarnitines-sialic acid-acetylated amino acids and amino acid derivatives: 2-Heptanone, 5′-Deoxy-5′-(methylthio)adenosine, cystathionine, hexanoylcarnitine, N-Acetylneuraminic acid, N-Acetyl-L-phenylalanine, 2,2′-Thiodiacetic acid, octanoylcarnitine, trisaccharides, hydroxyphenylacetic acids, N6,N6,N6-Trimethyl-L-lysine, N-Acetyl-L-alanine, N-Acetyl-Asp-Glu, N-Acetyl-L-tyrosine, butyrylcarnitine, p-Hydroxyphenyllactic acid, phenyIllactic acid, N-Acetyl-L-aspartic acid, L-Saccharopine, Methylimidazoleacetic acid and N-Formyl-L-methionine. Taken together, these metabolites are indicative that ACLF associates with increased proteolysis and lipolysis. As expected in the context of acute inflammation, several members of this group were amino acids or amino acid metabolites indicating increased proteolysis (highlighted by increased N6,N6,N6-trimethyl-L-lysine levels) and depression in mitochondrial β-oxidation and energy production acylcarnitines and N6,N6,N6-trimethyl-L-lysine—a lysine catabolite, precursor of carnitine.

[0036] Group 2: sugar alcohols, catecholamines and pyrimidine derivatives are pentose phosphates, pentose alcohols, hexose alcohols, D-Galacturonic acid, beta-Pseudouridine, D-glucuronic acid, 4-Hydroxy-3-methoxyphenylglycol sulfate, D-Threitol, mevalonic acid, orotidine, and Guanidinosuccinic acid. Taken together, these metabolites are indicative of changes in intracellular glucose utilization.

TABLE-US-00002 TABLE 2 Metabolites of the ACLF fingerprint belonging to Group 1. Acute Healthy Compensated Descompensation Acute-on-chronic Metabolites subjects (HS) Cirrhosis (CC) (AD) liver failure (ACLF) 2-Heptanone 10,032.86 2,949.65 13,336.89 129,161.02 5′-Deoxy-5′- 10,816.94 9,479.17 19,834.54 58,796.35 (methylthio)adenosine Cystathionine 6,858.25 13,620.37 34,138.99 105,726.39 Hexanoylcarnitine 117,700.74 115,320.79 176,519.30 382,225.20 N-Acetylneuraminic 117,782.77 117,247.16 175,998.67 396,867.29 acid N-Acetyl-L- 58,237.42 70,903.52 102,361.18 184,768.46 phenylalanine 2,2′-Thiodiacetic acid 31,393.88 48,127.67 62,461.24 164,988.82 Octanoylcarnitine 513,068.96 499,733.11 459,512.43 877,484.16 Trisaccharides 46,597.92 29,509.79 34,971.07 78,193.50 Hydroxyphenylacetic 126,924.40 222,894.02 327,445.02 1,678,736.63 acids N6,N6,N6-Trimethyl-L- 180,797.36 184,391.24 209,118.01 363,853.99 lysine N-Acetyl-L-alanine 2,079,926.52 2,276,785.18 2,815,879.27 5,340,423.80 N-Acetyl-Aps-Glu 10,445.86 6,833.99 8,762.63 27,944.13 N-Acetyl-L-tyrosine 44,566.33 60,586.55 93,667.18 224,228.24 Butyrylcarnitine 157,168.47 183,301.95 242,417.98 410,452.47 p-Hydroxyphenyllactic 4,994,470.91 8,325,683.99 13,880,240.53 26,698,885.54 acid Phenyllactic acid 413,797.83 500,460.67 1,310,988.98 3,659,154.88 N-Acetyl-L-aspartic 796,683.72 899,318.28 1,432,471.96 2,156,047.10 acid L-saccharopine 263.34 501.01 2,800.04 18,718.55 Methylimidazoleacetic 51,979.78 37,316.87 41,805.55 117,956.11 acid N-Formyl-L-methionine 589,849.73 641,460.25 839,940.74 1,448,062.27

TABLE-US-00003 TABLE 3 Metabolites of the ACLF fingerprint belonging to Group 2. Healthy Compensated Acute Acute-on-chronic subjects Cirrhosis Descompensation liver failure Metabolites (HS) (CC) (AD) (ACLF) Pentose phosphates 3,067.12 7,564.74 20,067.11 148,855.72 Pentose alcohols 530,205.16 561,146.65 969,767.29 2,491,598.60 Hexose alcohols 36,986.60 101,728.82 299,248.27 1,240,715.56 D-Galacturonic acid 993,841.27 1,115,891.33 4,443,815.93 17,114,085.26 beta-Pseudouridine 2,176,139.87 2,596,211.16 3,230,664.86 7,394,309.37 D-glucuronic acid 876,065.68 711,537.00 1,617,539.49 3,702,563.66 4-Hydroxy-3- 33,443.40 64,691.63 122,598.79 343,222.58 methoxyphenylglycol sulfate D-Threitol 678,230.62 725,307.54 1,152,202.95) 4,161,860.27 Mevalonic acid 197,284.72 164,678.55 215,300.35 695,399.34 Orotidine 7,697.22 43,876.28 57,554.13 214,329.76 Guanidinosuccinic 19,940.20 9,928.06 6,683.85 54,470.69 acid

Example 3: Characterization of Patient's Metabolic Profile Associated with Organ Failure.

[0037] The aim of the study was also to characterize the metabolic profile associated with each of the three main organ failures (liver, brain, and kidney) and to identify potential pathophysiological mechanisms of the syndrome.

[0038] The results shows that the intensity of the ACLF-associated fingerprint increased across ACLF grades, and was similar in patients with kidney failure and in those without, indicating that it was not a simple reflect of decreased kidney excretion but also of cell metabolism alterations.

TABLE-US-00004 TABLE 4 Group 1: acylcarnitines-sialic acid-acetylated amino acids and amino acid derivatives associated with organ failure. Metabolites Liver Brain Kidney 2-Heptanone 7,494.78 13,213.92 47,048.72 5′-Deoxy-5′- 24,227.16 16,681.46 26,321.10 (methylthio)adenosine Cystathionine 41,900.94 28,850.60 60,393.41 Hexanoylcarnitine 187,933.79 152,359.55 314,654.54 N-Acetylneuraminic 177,728.48 162,372.55 425,377.27 acid N-Acetyl-L- 120,035.36 89,777.65 142,744.45 phenylalanine 2,2′-Thiodiacetic 63,675.66 56,727.91 131,258.98 acid Octanoylcarnitine 517,116.54 375,421.77 843,573.43 Trisaccharides 30,126.10 37,284.86 58,879.75 Hydroxyphenylacetic 299,940.71 414,336.25 1,244,165.22 acids N6,N6,N6-Trimethyl- 185,403.12 209,872.91 362,158.61 L-lysine N-Acetyl-L-alanine 3,032,014.88 2,718,874.31 4,642,470.87 N-Acetyl-Aps-Glu 9,689.51 7,827.76 19,555.43 N-Acetyl-L-tyrosine 174,755.17 87,314.29 101,194.46 Butyrylcarnitine 229,533.80 232,083.14 331,550.94 p-Hydroxy- 16,542,518.37 13,974,711.59 18,065,569.79 phenyllactic acid Phenyllactic acid 1,597,135.70 1,325,688.29 1,941,754.79 N-Acetyl-L- 1,449,118.53 1,408,189.24 1,859,825.40 aspartic acid L-saccharopine 3,821.89 2,352.30 9,529.67 Methyl- 37,376.40 43,503.77 222,579.87 imidazoleacetic acid N-Formyl-L- 927,280.10 783,272.67 1,202,024.57 methionine

TABLE-US-00005 TABLE 5 Group 2: sugar alcohols, catecholamines and pyrimidine derivatives associated with organ failure. Metabolites Liver Brain Kidney Pentose phosphates 73,109.05 15,965.82 64,254.82 Pentose alcohols 1,006,221.30 896,205.74 2,456,859.506 Hexose alcohols 288,326.48 353,578.42 1,095,887.94 D-Galacturonic acid 15,702,997.27 3,273,384.07 9,230,861.51 beta-Pseudouridine 3,322,371.50 3,004,062.61 6,785,670.06 D-glucuronic acid 3,697,356.71 1,409,747.45 2,134,268.17 4-Hydroxy-3- 116,363.21 120,989.45 233,736.82 methoxyphenylglycol sulfate D-Threitol 933,354.55 1,235,143.88 4,518,066.25 Mevalonic acid 179,786.24 194,641.41 736,822.80 Orotidine 50,462.09 55,267.09 253,315.85 Guanidinosuccinic acid 2,161.95 6,416.88 63,689.84

[0039] According to these data, ACLF can be distinguishable from AD by a blood metabolite fingerprint capturing blood metabolite accumulation in ACLF and witnessing profound multiorgan alterations in major metabolic pathways, in particular depressed energy production, which may partake into the development of organ failures. In addition, metabolites accumulated in blood may contribute to the severity via toxic effects.